Our long-term objective is to understand signaling pathways that protect the heart from ischemia/reperfusion (I/R) injury. Our short-term objective, and the topic of this proposal, is to examine the mechanism of cardioprotection by the cytosolic small heat shock protein (sHSP), alphaB-crystallin (alphaBC). alphaBC (22 kDa) is expressed predominantly in cell types with high oxidative phosphorylation demands, e.g. cardiac myocytes. In cardiac myocytes, alphaBC is phosphorylated upon stimulation of stress MAP kinases (e.g. p38), which, if chronically activated, also drive increased alphaBC expression. Increasing the level of wt alphaBC, or expressing pseudophosphorylated alphaBC, protects against I/R injury. Our preliminary data indicate that mitochondria serve as an alphaBC-binding target during I/R. Accordingly, our hypothesis is that phosphorylated alphaBC protects cardiac myocytes from I/R injury and a portion of this protection is mediated by the conditional association of phospho-alphaBC with mitochondrial outer membrane (mOM) proteins, such as the MPTP. The Specific Aims that address this hypothesis are to: 1) examine the kinetics with which I or I/R increase m-alphaBC, and to assess the phosphorylation status of m-alphaBC, using a combination of immunoblotting, confocal and electron microscopy, 2) assess the effects of alphaBC deletion, or overexpression of wild type or mutant forms of alphaBC on MPTP activation, apoptosis, autophagy and myocardial function in response to I/R, and 3) identify mitochondrial alphaBC binding partners and elucidate l/R-dependent changes in the mitochondrial subproteome. Significance and Innovation: The proposed studies are the first to examine the interaction of alphaBC with mitochondria in any tissue type, and the first to study how m-alphaBC preserves mitochondrial function during stress. These studies employ a comprehensive series of experiments using state-of-the-art cellular, molecular genetic and proteomics technologies to discover new information required to understand cellular mechanisms that preserve mitochondrial function during I/R stress.